ABSTRACT Commensalbacterialpopulationsintheoralcavitynotonlyplayapivotalroleinthemaintenanceofhealthy oralmucosa,butalsomakesforattractivetargetsforinducedsecretionoftherapeuticmolecules,suchas peptide/proteintherapeuticsorimmunogensfororalvaccines.Studieshaveshownthatgeneticallyengineered commensalsinoculatedingermfreeanimalscanproducetherapeuticallyrelevantproteins,suchas immunogensthatelicitmucosalantibodyproduction.However,clinicaltranslationofthesetheselivebacterial therapiesfacesmanyhurdles,including(i)difficultyindisplacingexistingcommensalpopulationswiththe engineeredvariants,(ii)safetyconcernswiththeengineeredvariants,and(iii)characterization,storageand handlingoflivebacteria.Withtheexceptionoffecaltransplantsinpatientswhohavereceivedextensive antibioticstherapy,nolivebacteriaarecurrentlyusedasatherapyinclinicalsetting.Inthisproposal,Iseekto developanalternativestrategythatallowsdirectmodificationofexistingcommensalpopulations.Specifically, Iwillengineerbacterialviruses,orbacteriophages,togeneticallymodifycommensalbacterialpopulationsat mucosalsurfaces?insitu?.Bacteriophagespresentnohumantoxicityorpathogenicity,andareefficient transductionvectorswithhighspecificity.However,todate,virtuallyallbacteriophagedevelopmentfocuses onusing?lyticphages?tokillspecificpathogenicbacteria(i.e.bacteriophagesasanewclassofantimicrobial), andlittleworkhasbeendoneonengineeringtemperate? ?phagestomodifytheproteinexpressionandsecretion profilesofcommensalbacteria.TheprimaryresearchgoalofthisF32traininggrantistodemonstratethe proofofconceptthatengineeredphagescanmediateefficienttransferofgeneticmaterialtocommensal bacteria.InAim1,IwillisolatethreetemperatebacteriophagefromdifferentS? treptococcusMitis?strains, introduceareportergene(TagRFP)intoitsgenome,andassesstheirpotencyintransducingS? .Mitis?isolated fromhumansalivaviaflowcytometry.Aim2extendsthismethodtothedisplayandsecretionofmodel therapeuticmolecules.Usingthemostpotentbacteriophagevector,Iwillincorporatemodelproteinslinkedto bacterialsecretion/displaytags,andmeasuretheextentthattheseproteinswillbesecretedbyordisplayedon thesamehumanderived?S.mitis?.InAim3,usinggnotobioticmiceinoculatedwithhumanS? .mitis?,Iwill quantifytheamountofsecretedproteinintheoralandgastrointestinalmucosainducedbyengineeredphage particles,aswellasantibodyresponsetoproteinspresentedonthesurfaceoftransduced?S.mitis?.Theresults willprovideimportantinsightsintothepotentialuseoftemperatephagestomodifycommensalpopulationsfor deliveryofspecificproteintherapiesandimmunogens.